Birch bark processing and the isolation of natural products from birch bark

ABSTRACT

The invention provides methods for separating outer birch bark from inner birch bark. The invention also provides methods for isolating betulin; lupeol; betulinic acid; 9,10-epoxy-18-hydroxyoctadecanoic acid; 9,10,18-trihydroxyoctadecanoic acid; polyphenolic polymers and fatty acids from birch bark.

BACKGROUND OF THE INVENTION

Birch bark is a low-value waste product in the forest industry today.Ekman, R., Holzforschung, (1983) 37, 205. Approximately 230,000 tons ofbirch bark are generated per year. For example, a single paper mill cangenerate 70 tons of birch bark per day. Thus, vast quantities of birchbark and its chemical components are available.

Birch bark is a potential source of a variety of organic chemicals.Several triterpenoids have been identified in birch bark extracts. Forexample, lupeol, betulin, betulinic aldehyde, betulinic acid, methylbetulinate, lupenone, betulonic aldehyde, betulonic acid, β-amyrin,erythrodiol, oleanolic aldehyde, oleanolic acid, methyl leanolate andacetyl oleanolic acid are all present in the outer bark of Betulaverrucosa. Eckerman, C., (1985) Paperi ja Puu, No. 3, 100. In addition,several suberinic acids isolated from birch bark, as well as severaltriterpenoids, have been identified in the bark of Betula verrucosa.Ekman, R., Holzforschng, (1983) 37, 205.

The chemical constituents of birch bark are useful in pharmaceutical andindustrial applications. For example, U.S. Pat. No. 5,750,578 disclosesthat betulin possesses antiviral properties and is useful to treatherpesvirus. Betulin also possesses antifeedant activity against bollweevils, and anti-inflammatory activity (Miles, D. H., 1994, J. Agric.Food. Chem., 42, 1561-1562 and Recio, M., Planta Med., 1995, 61, 9-12.In addition, betulin showed cough suppressant and expectorant effects.Jinuhua, W., Zhongguo Yaoxue Zazhi, (1994), 29(5), 268-71. Betulin isalso a useful starting material for preparing alobetulin and derivativesthereof, which posses useful pharmacological properties.

Betulin can be converted to betulinic acid, which is useful as atherapeutic agent. For example, Pisha, E. et al., (1995) J. M. NatureMedicine, 1, 1046-1051 discloses that betulinic acid has antitumoractivity against human melanoma, e.g., MEL-1, MEL-2 and MEL-4. Inaddition, Fujioka, T. et al., J. Nat. Prod., (1994) 57, 243-247discloses that betulinic acid has anti-HIV activity in H9 lymphocyticcells.

Ambrettolide (cis-hexadec-7-enolide), a naturally occurring compound, isused to induce musk fragrance in perfumes. Ambrettolide is found in thevegetable oil of ambrette seeds. The synthesis of ambrettolide isaccomplished from 9,10,18-trihydroxyoctadecanoic acid via ahigh-yielding multi-step synthesis. Seoane, E., J. Chem. Soc. PerkinTrans. (1982), 1837-1839. Therefore, 9,10,18-trihydroxyoctadecanoicacid, which is present in birch bark, is a useful precursor for thesynthesis of ambrettolide.

9,10-Epoxy-1 8-hydroxyoctadecanoic acid is also present in birch bark.9,10-Epoxy-18-hydroxyoctadecanoic acid is an environmentally-friendlyspoilage deterrent and a rot-resistant additive for wood composites.Sweitzer, P., et al., Induction of Resistance in Barley Against Erysiphegraminis by Free Cutin Monomers, Physiol. Mol. Plant Pathol, (1996)49(2) 103-120.

Suberin is another major component of birch bark. Suberin is aninsoluble polymeric material that is attached to the cell walls ofperiderms. Kola, P. E. et al., Ann. Rev. Plant. Physiol., (1981), 32:539-67. Suberin is generally an ester of fatty acids and polyphenolicpolymers. Suberin of birch bark is typically a biopolyester of primaryhydroxy, epoxy and dicarboxylic acids. Ekman, Holzforschung, (1983) 37,205-211.

Suberin posseses several industrial applications. See, e.g., Taylor andFrancis, Forests Products Biotechnology, A. Bruce and J. W. Palfreyman(editors), 167, 179-181 (199); Peter E. Laks and Peggy A. McKaig,Flavonoid Biocides: Wood Preservatives Based on Condensed Tannins,Horzfschung, 42, 299-306 (1988); Etherington & Roberts,Dictionary—birch(bark),http://sul-server2.stanford.edu/don/dt/dt0328.html, 1, Jun. 23, 1999; P.E. Kolattukudy, Structure, Biosynthesis, and Biodegradation of Cutin andSuberin, Ann. Rev. Plant Physiol., 32, 539-67 (1981); and N. Cordeiro,M. N. Belgasem, A. J. D. Silvestre, C. Pascol Neto, A. Gandini, CorkSuberin as a new source of chemicals, Int. Journal of BiologicalMaterials, 22, 71080 (1998). Suberin is useful as a dispersant in manyindustrial applications (e.g., carbon black slurries, clay products,dyes, cement, oil drilling muds, and asphalt emulsifiers). Suberin isalso useful in binders for animal pellets, conditioners for boilingwater, anti-oxidants and additives to lead-storage battery plateexpanders. McGraw-Hill Concise Encyclopedia of Science & Technology,Fourth Edition, 1998.

Polyphenolic polymers are also present in birch bark as a constituent ofsuberin. Polyphenolic polymers may be classified as soluble polyphenolicpolymers or non-soluble polyphenolic polymers. Soluble polyphenolicpolymers are the portion of polymers which are soluble in water underboth acidic and basic conditions. The non-soluble polyphenolic polymersare non-soluble in water at a pH below about 4.0, but soluble inacetone, alcohols and other polar solvents. The non-soluble polyphenolicpolymers may have a different formulation from the soluble polyphenolicpolymers. However, the non-soluble polyphenolic polymers may be used inthe same industrial applications as the soluble polyphenolic polymers.

Polyphenolic polymers are non-toxic and biodegradable and may beformulated for numerous purposes (e.g., as anti-oxidant reagents,anti-fungal materials, coating materials, co-polymers, woodpreservatives, tire cord adhesives, foundry cord binders, rigid andfloral foams, ion exchange resins, industrial water purificationflocculants, textile dyes, food additives and pharmaceuticals). Pizzi,Wood Bark Extracts as Adhesives and Preservatives, 167-181, Taylor &Frances, Forest Products Biotechnology, Bruce and Palfreyman (editors),1998.

Current methods for isolating the chemical constituents of birch barkare deficient in several ways. For example, betulin has been extractedfrom the bark of white-barked birches in amounts up to 30%, based on thedry weight of the bark. Elkman, R., (1983) Holzforsch, 37, 205; Ohara,S., et al., (1986) Mokuza Gakkaishi, 32, 266. In addition, Betulin hasbeen isolated from outer birch bark waste of Betula verrucosa by liquidextraction employing boiling organic solvents and subsequentrecrystallization. Eckerman, C., (1985) Paperi ja Puu, No. 3, 100. Whilecurrent processes afford acceptable yields of betulin (e.g., 11-30%),these processes suffer from several major drawbacks. For example, theuse of a boiling organic solvent, at standard pressure, in theextraction of betulin may destroy other useful compounds present in thebark. A need therefore exists for a method that can be used to extractbetulin without damaging other compounds remaining in the birch bark.

Another drawback with the current extraction processes is that theorganic solvents employed are hazardous, difficult to handle ordifficult to dispose of. The typical organic solvents, which includemethylene chloride and chloroform, are hazardous to humans (i.e., theyare toxic or carcinogenic) and are hazardous to the environment.Considering the industrial scale on which the extraction processes wouldneed to be performed in order to provide industrial quantities (e.g.,tons) of betulin, large quantities of organic solvents would berequired. The high cost of disposing the organic solvents is anadditional disadvantage of the current extraction procedures.

Several methods have been devised for isolating polyphenolic polymersfrom birch trees. Some isolation methods are based on acid treatments inwhich the carbohydrate components (cellulose and hemicelluloses) arehydrolyzed to water-soluble materials. However, with such procedures,serious doubts exist as to whether the isolated polyphenolic polymer isrepresentative of the “native” polyphenolic polymer. In addition,extraction conditions can cause undesirable rearrangements and othertransformations of the polyphenolic structure that lead to a loss ofuseful properties. It is therefore desirable to have polyphenolicpolymers in a form in which it is readily accessible, without involvingcostly, lengthy or dangerous procedures.

Suberin from betula verucosa contains at least 35 fatty acids whichmakes it hardly usable in industry. U.S. Pat. No. 4,732,708 issued toEkman, R. et al. discloses a process for manufacturing suberinic acid.The process, however, does not attempt to separate the individual fattyacids. In addition, due to the crucial differences in the fundamentalchemistry between the types of birch trees (i.e., the type anddistribution of fatty acids), the procedures employed in U.S. Pat. No.4,732,708 issued to Ekman, R. et al. may not be useful for the isolationof fatty acids from species of birch bark other than those employed inU.S. Pat. No. 4,732,708. As such, a method for isolating the individualfatty acids from the bark of other species of birch is needed.

The current methods employed to isolate not only betulin, but othercomponents in birch bark (e.g., lupeol; betulinic acid;9,10-epoxy-18-hydroxyoctadecanoic acid; 9,10,18-trihydroxyoctadecanoicacid; and polyphenolic polymers) are costly, inefficient or unsafe. Aneed therefore exists for safer, more cost-efficient methods to obtaincommercial quantities (e.g., tons) of betulin; as well as commercialquantities (e.g., kg) of lupeol; betulinic acid;9,10-epoxy-18-hydroxyoctadecanoic acid; 9,10,18-trihydroxyoctadecanoicacid; and polyphenolic polymers from birch bark. In addition, a needalso exists for an industrial scale process for producing theseproducts.

SUMMARY OF THE INVENTION

The present invention provides methods for isolating the chemicalconstituents of birch bark. Specifically, the present invention providesa method that can be used to extract betulin from birch bark withoutdamaging other compounds remaining in the birch bark. In addition, theextraction processes employ solvents that are safe (non-toxic andnon-carcinogenic), easy to handle, environmentally-friendly,inexpensive, and easy to dispose of. The present invention also providesa method for isolating polyphenolic polymers from birch trees whereinthe polyphenolic polymers are in a form that is readily accessible andthe methods do not involve costly, lengthy or dangerous procedures. Thepresent invention also provides a method for isolating the individualfatty acids from the bark of various species of birch. The presentinvention also provides methods to provide commercial quantities (e.g.,tons) of betulin; as well as commercial quantities (e.g., kg) of lupeol;betulinic acid; 9,10-epoxy-18-hydroxyoctadecanoic acid;9,10,18-trihydroxyoctadecanoic acid; and polyphenolic polymers frombirch bark.

The present invention provides a process for separating outer birch barkfrom inner birch bark comprising subjecting birch bark to fragmentationto provide a combination of outer birch bark shreds and inner birch barkchunks and separating the outer birch bark shreds from the inner birchbark chunks.

The present invention also provides a process that provides one or more(e.g., 1, 2, 3, or 4) natural products from outer birch bark.Accordingly, there is provided a process for obtaining one or morenatural products from outer birch bark comprising subjecting the outerbirch bark to supercritical fluid extraction to provide the naturalproduct.

The present invention also provides a process for obtaining lupeol,betulinic acid and betulin from outer birch bark comprising extractingouter birch bark with carbon dioxide at a pressure between about 3,000psi and 10,000 psi and at a temperature between about 50° C. and 100° C.to provide lupeol, betulin and betulinic acid.

The present invention also provides a process for obtaining lupeol,betulinic acid and betulin from outer birch bark using fractionalsupercritical fluid extraction comprising extracting with carbon dioxideat a pressure below about 5,000 psi and at a temperature below about 50°C. to provide a product comprising lupeol and extracting with carbondioxide at a pressure of about 5,000 psi to about 10,000 psi and at atemperature of about 50° C. to about 120° C. to provide a productcomprising a mixture of betulin and betulinic acid.

The present invention also provides a process for obtaining lupeol fromouter birch bark comprising subjecting the outer birch bark tosupercritical fluid extraction with carbon dioxide at a temperature ofabout 40° C. to about 50° C. and a pressure of about 3,000 psi to about5,000 psi for a period of time of about 1 hour to about 3 hours toprovide the lupeol.

The present invention also provides a process for obtaining betulin andbetulinic acid from outer birch bark comprising subjecting the outerbirch bark to supercritical fluid extraction with carbon dioxide at atemperature of about 80° C. to about 100° C. and a pressure of about8,000 psi to about 10,000 psi for a period of time of about 3 hours toabout 5 hours to provide a mixture of betulin and betulinic acid.

The present invention also provides a process for isolating9,10-epoxy-18-hydroxyoctadecanoic acid from outer birch bark comprising:(1) subjecting the outer birch bark to alkali hydrolysis in an aqueousalcohol solution to provide a second outer birch bark and a secondsolution; (2) separating the second solution from the second outer birchbark; (3) condensing the second solution at a temperature below about50° C. to form a third solution; (4) adding water to the third solutionto form a precipitate and a fourth solution; (5) separating theprecipitate from the fourth solution; (6) acidifying the fourth solutionto a pH of about 5.5 to about 6.5 to give a fifth solution and9,10-epoxy-18-hydroxydecanoic acid as a precipitate; and (7) separatingthe 9,10-epoxy-18-hydroxydecanoic acid precipitate from the fifthsolution to give 9,10-epoxy-18-hydroxydecanoic acid.

The present invention also provides a process for isolating9,10,18-trihydroxyoctadecanoic acid from outer birch bark comprising:(1) subjecting the outer birch bark to alkali hydrolysis in an aqueousalcohol solution to provide a second outer birch bark and a secondsolution; (2) separating the second solution from the second outer birchbark; (3) condensing the second solution at a temperature below about50° C. to form a third solution; (4) adding water to the third solutionto form a first precipitate and a fourth solution; (5) separating thefirst precipitate from the fourth solution; (6) acidifying the fourthsolution to a pH of about 5.5 to about 6.5 to give a fifth solution anda second precipitate; (7) separating the second precipitate from thefifth solution; (8) condensing the fifth solution to provide a sixthsolution; (9) subjecting the sixth solution to epoxidizing conditions toprovide an epoxide and hydrolyzing the epoxide to provide a seventhsolution; and (10) crystallizing the seventh solution to give9,10,18-trihydroxyoctadecanoic acid.

The present invention also provides a process for isolating non-solublepolyphenolic polymers and fatty acids from outer birch bark comprising:(1) subjecting the outer birch bark to alkali hydrolysis in an aqueousalcohol solution to provide a second birch bark and a second solution;(2) separating the second solution from the second outer birch bark; (3)adding water to the second outer birch bark to provide a third solutionand a third outer birch bark; (4) separating the third solution from thethird outer birch bark; (5) acidifying the third solution to a pH ofabout 3.0 to about 4.0 to give a fourth solution and a mixture ofnon-soluble polyphenolic polymer and fatty acids; and (6) separating themixture of fatty acids and non-soluble polyphenolic polymers from thefourth solution.

The present invention also provides a process for isolating fatty acidsand soluble polyphenolic polymers from outer birch bark comprising: (1)subjecting the outer birch bark to alkali hydrolysis in an aqueousalcohol solution to provide a second outer birch bark and a secondsolution; (2) separating the second solution from the second outer birchbark; (3) adding water to the second outer birch bark to provide a thirdouter birch bark and a third solution; (4) separating the third solutionfrom the third outer birch bark; (5) acidifying the third solution to apH of about 3.04-4.0 to give a fourth solution and a solid; (6)separating the solid from the fourth solution; (7) adding an alcohol tothe fourth solution to provide a fifth solution and a precipitate; (8)separating the precipitate from the fifth solution; and (9) condensingthe fifth solution to provide a mixture of fatty acids and solublepolyphenolic polymers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(a) illustrates outer and inner birch bark (cross-sectional view).

FIG. 1 (b) illustrates outer and inner birch bark.

FIG. 2 illustrates an apparatus for supercritical fluid extraction.

FIG. 3 is a schematic illustration of the isolation of lupeol, betulinand betulinic acid from outer birch bark.

FIG. 4 is a schematic illustration of the isolation of9,10-epoxy-18-hydroxyoctadecanoic acid from outer birch bark.

FIG. 5 is a schematic illustration of the isolation of9,10,18-trihydroxyoctadecanoic acid from outer birch bark.

FIG. 6 is a schematic illustration of the isolation of a mixture offatty acids and non-soluble polyphenolic polymers (NPPP) from outerbirch bark.

FIG. 7 is a schematic illustration of the isolation of a mixture offatty acids and soluble polyphenolic polymers (SPPP) from outer birchbark.

FIG. 8 is a schematic drawing of the isolation of lupeol; betulin;betulinic acid; 9,10-epoxy-18-hydroxyoctadecanoic acid;9,10,18-trihydroxyoctadecanoic acid; a mixture of fatty acids andnon-soluble polyphenolic polymers (NPPP); and a mixture of fatty acidsand soluble polyphenolic polymers (SPPP) from outer birch bark.

FIG. 9 illustrates the separation of outer and inner bark with an airclassifier.

DETAILED DESCRIPTION OF THE INVENTION

Specific values listed below for ranges are for illustration only; theydo not exclude other defined values or other values within definedranges.

Separation of Inner Birch Bark from Outer Birch Bark

As used herein, “birch” is any of the several deciduous trees of thegenus Betula. The birches comprise the family Betulaceae in the orderFagales. Birch trees include, for example, white birch, B. alba; sweet,black or cherry birch, B. lenta; monarch birch, B. maximowicziana; dwarfor arctic birch, B. nana; Japanese white birch, B. platyphyla japonica;smooth-bark birch, B. pubescens; yellow birch, B. alleghaniensis; paper,white or canoe birch, B. papyrifera; grey birch, B. populifolia; riverbirch, B. nigra; and the European birches, B. pubescens; B. alba and B.pendula. Specifically, birch can be B. alba, B. lenta, B.maximowicziana, B. nana, B. platyphyla japonica, B. pubescens, B.alleghaniensis, B. papyrifera, B. populifolia, B. nigra, B. pubescens,B. alba or B. pendula. A specific birch for use in the processes of thepresent invention is B. papyrifera.

As used herein, “fragmentation” includes chopping, crunching, crushing,gnashing or pounding. Such fragmentation of birch bark will effectivelyprovide inner birch bark (e.g., in the form of chunks) and outer birchbark (e.g., in the form of shreds) which can be physically separated.The fragmentation can conveniently be carried out by feeding birch barkinto a machine with knives on a rotating disk (e.g., a chipper orshredder). One chipper suitable for fragmenting the bark is the YardManModel 246-648D401 chipper.

As illustrated in FIG. 1 (a) and FIG. 1 (b), birch bark consists ofinner birch bark and outer birch bark. Inner birch bark is more denseand granular than outer birch bark, while outer birch bark is moreflexible and fibrous than inner birch bark. In addition, outer birchbark is light in color, thin (1-5 mm), tough, and of low water-contentrelative to inner birch bark. The inner bark is darker in color, thicker(3-10 mm) and non-fibrous relative to the outer bark. The inner bark isthe portion of the tree wherein significant water transport occurs(i.e., an area of high water content). Due to the differences in thephysical properties of inner birch bark and outer birch bark, Applicanthas found that fragmentation produces outer birch bark shreds and innerbirch bark chunks.

Outer birch bark shreds can be separated from the inner birch barkchunks using any suitable means. The separation can conveniently beaccomplished by screening the mixture through a mesh having openingsintermediate in size between the smaller inner bark chunks and thelarger outer bark shreds. The smaller inner bark chunks fall through thescreen and are separated from the outer bark.

The “mesh” can be a unit comprising one or more open spaces in a cord,thread, or wire network in which the cords, threads or wires surroundthe spaces. Any mesh suitable to separate inner birch bark from outerbirch bark can be employed. Typically, the mesh is a wire meshcontaining openings of about ½ of an inch by ½ of an inch, or smaller.

For example, mesh can conveniently contain openings of about ¼ of aninch by about ¼ of an inch. Specifically, the size of the mesh can beabout 20 mm by about 20 mm, or about 10 mm by about 10 mm, or about 6 mmby about 6 mm. More specifically, the size of the mesh can be about 3 mmby about 3 mm.

Alternatively, the inner birch bark chunks and outer birch bark shredsmay be separated with the use of an air classifier as shown in FIG. 9.As used herein, an “air classifier” is a device which operates on theprinciple of the differing properties of the two components (e.g., innerand outer birch bark) in an air stream to effect a physical separation.Typically, the less dense outer bark travels a greater distance in theair stream than the more dense inner bark. The inner bark, along withother materials, falls rapidly from the stream of air. As a result, theinner birch bark and the outer birch bark can be separated.

After separating outer birch bark from inner birch bark, outer birchbark of about 10 wt. % to about 45 wt. % based on initial birch barkcontent is typically obtained and inner birch bark of about 55 wt. % toabout 85 wt. % is typically obtained.

For use in the processes of the present invention, birch bark shredsless than about 10 mm in diameter can conveniently be used. Morespecifically, outer birch bark shreds less than about 6 mm in diameter,less than about 4 mm in diameter, or less than about 2 mm in diameter,can be used.

Supercritical Fluid Extraction

As illustrated in FIG. 2, a natural product (e.g., betulin, betulinicacid or lupeol) can be obtained from outer birch bark by supercriticalfluid extraction. The outer birch bark is introduced into a feed tank(1) through the opened lid on the top. The birch bark is heated at anelevated pressure in a solvent comprising carbon dioxide. The solutionis transferred to a product reservoir (2). Extracted product is removedand the solvent comprising carbon dioxide is passed though a condenser(3) and subsequently recycled into the feed tank (1) through a recycler(4).

As used herein, “natural product” is any of the compounds naturallyoccurring in the bark of birch. Natural products specifically includetriterpenoids.

According to the biogenetic isoprene rule, a “triterpenoid” is ahydrocarbon, or its oxygenated analog, that is derived from squalene bya sequence of straightforward cyclizations, functionalizations, andsometimes rearrangement.

A specific triterpenoid present in birch bark is betulin(lup-20(29)ene-3,28-diol), lupeol (lup-20(29)-en-3β-ol), or betulinicacid (lup-20(29)-en-3β-ol-28-oic acid).

The processes of the present invention provide natural products of birchbark. Each natural product may have one or more chiral centers and mayexist in and be isolated in optically active and racemic forms. It is tobe understood that the present invention provides processes forisolating natural products in any racemic, optically-active,polymorphic, or stereoisomeric form, present in the native bark orisolated after exposure to the processes of the invention. When aprocess of the invention provides a mixture of enantiomers or isomers,it is appreciated that those skilled in the art can separate opticallyactive forms (for example, by resolution of the racemic form byrecrystallization techniques or by chromatographic separation using achiral stationary phase) if a single enantiomer is desired.

Supercritical fluid extraction is an extraction wherein a fluid at atemperature and pressure above its critical point is employed; or afluid above its critical temperature, regardless of pressure, isemployed. Below the critical point, the fluid can coexist in both gasand liquid phases, but above the critical point there is only one phase.Equipment and techniques for carrying out supercritical fluid extractionare known to those skilled in the art. See, McHugh, M. And Krukonis, V.,Supercritical Fluid Extraction, 2nd ed, Butterworth-Heinemann, Boston,1994; Johnston, K. P., Penninger, J. M. L., Supercritical Fluid Scienceand Technology, ACS Symposium Series 406, American Chemical Society,Washington, D.C.; and Taylor, L. T., Supercritical Fluid Extraction,John Wiley & Sons, New York, 1996.

In a supercritical fluid extraction, thermodynamic and transportproperties of supercritical fluid are a function of density, whichdepends strongly on the fluid's pressure and temperature. The densitymay be adjusted from a gas-like value of 0.1 g/ml to a liquid-like valueas high as 1.2 g/ml. Furthermore, as conditions approach the criticalpoint, the effect of temperature and pressure on density becomes muchmore significant. For example, increasing the density of supercriticalcarbon dioxide from 0.2 to 0.5 g/ml requires raising the pressure from85 atm to 140 atm (8.6 megapascals to 14.2 megapascals) at 158° F. (70°C.), but at 95° F. (35° C.) the required change is only from 65 atm to80 atm (6.61 Mpa to 8.1 Mpa).

As used herein, “fractional supercritical fluid extraction” (hereinafter“FSCFE”) is a multi-step procedure wherein the supercritical fluidextraction is carried out at one temperature and pressure for a givenperiod of time and is then carried out at one or more other temperaturesor pressures.

The efficiency of supercritical fluid extraction on a material such asouter birch bark depends in part upon the size of the outer birch barkpieces. Thus, the smaller the outer birch bark pieces, the moreefficient the supercritical fluid extraction typically will be. As such,after fragmentation and prior to extraction, outer birch bark shreds maybe further reduced in size with a Hammermill or suitable means. Forexample, a 15 horsepower 3B Junior Hammermill made by Jay BeeManufacturing, Inc can be used as illustrated in the Examples hereinbelow. The hammermill reduces large pieces of birch bark by beating thebark with pivoted hammers until the material is small enough to fallthrough a mesh.

For use in the processes of the present invention, the size of outerbirch bark shreds obtained after the Hammermill reduction is typicallyless than about 5 mm in diameter. Specifically, the shreds can be lessthan about 3 mm in diameter. More specifically, the shreds can be lessthan about 1 mm in diameter.

Prior to fragmentation or extraction, outer birch bark may be dried ofany water present. Such drying may increase the efficiency of thefragmentation. Birch bark may be air-dried or dried at an elevatedtemperature with or without reduced pressure (i.e., in vacuo).Specifically, birch bark may be dried in vacuo at an elevatedtemperature. Machines capable of drying bark are known in the art andinclude an oven, or similar device, such as a rotating air drum drier.

For use in the processes of the present invention, supercritical fluidextraction can conveniently be carried out at a pressure of about 1,000psi to about 12,000 psi. It is appreciated that those skilled in the artunderstand that higher pressures may enable faster extraction. In thiscase, it may be necessary to subsequently separate and purify theproduct (e.g., lupeol, betulin, betulinic acids, other minor triterpeneor acidic admixtures).

For use in the processes of the present invention, supercritical fluidextraction can conveniently be carried out at a pressure of about 750psi to about 12,000 psi. Specifically, the pressure may be about 1,000psi to about 10,000 psi. More specifically, the pressure may be about4,000 psi to about 9,000 psi.

For use in the processes of the present invention, the temperature atwhich the birch bark may be dried is greater than about 30° C.Specifically, the temperature is greater than about 45° C. Morespecifically, the temperature is greater than about 60° C.

For use in the processes of the present invention, the temperature ofsupercritical fluid extraction can conveniently be about 0° C. to about150° C. Specifically, the temperature can be about 25° C. to about 110°C. More specifically, the temperature can be about 45° C. to about 100°C.

In one specific embodiment, supercritical fluid extraction is performedat a temperature of about 40° C. to about 90° C. and a pressure of about3,000 psi to about 10,000 psi.

Supercritical fluid extraction employs a solvent which possessesphysical properties suitable as a supercritical fluid. Suitable solventsinclude carbon dioxide, Xe, Freon-23, ethane, N₂O, SF₆, propane,ammonia, n-C₄H₁₀, (C₂H₅)₂O and the like.

The physical properties of carbon dioxide make it particularlyattractive as a solvent for supercritical fluid extraction. Carbondioxide is a major component of the atmosphere and is thereforerelatively safe and abundant. In addition, carbon dioxide is relativelyinexpensive. Compared to most other suitable solvents, carbon dioxide isenvironmentally friendly as it will not harm the atmosphere at thequantities used in the methods of the invention. Moreover, carbondioxide is non-flammable and non-explosive. Further, carbon dioxideleaves no substantial residue or remnant upon evaporation.

Carbon dioxide also possesses physical properties which enable it tochange polarity over the temperature range and pressure range normallyemployed in supercritical fluid extraction. As a result, carbon dioxidemay act as a nonpolar solvent at one temperature and pressure but mayact as a polar solvent at another temperature and pressure. By varyingthe temperature and pressure, the solvent properties may be modified.This allows for the isolation of more than one compound using a singlesolvent system.

The solvent employed in supercritical fluid extraction may be a singlecompound or may be a mixture of compounds. In addition, the solvent mayinclude an additive.

As used herein, an “additive” is a compound added to the solvent in anamount of about 1 wt % to about 20 wt. % based on the solvent.Specifically, the additive may be present in an amount of about 1 wt. %to about 15 wt. % or about 1 wt. % to about 10 wt. %. Upon addition, theadditive will modify the physical properties of the solvent. Forexample, an additive may be useful to modify the polarity, criticaltemperature, critical pressure, etc., of the solvent system.

Suitable additives include lower alcohols (e.g., methanol, ethanol,1-propanol, 2-propanol, 1-hexanol, or 2-methoxy ethanol); ethers (e.g.,tetrahydrofuran or 1,4-dioxane); substituted hydrocarbons (e.g.,acetonitrile, dichloromethane, ammonia or chloroform) propylenecarbonate, N,N-dimethylaceamide; dimethyl sulfoxide; carboxylic acids(e.g., formic acid); water; carbon disulfide; lower ketones (e.g.,acetone), hydrocarbons (e.g., propane, toluene, hexanes and pentanes).

Applicant has found that utilizing a solvent comprising carbon dioxidein supercritical fluid extraction, lupeol is soluble at a temperaturebelow about 50° C. and a pressure below about 5,000 psi. In addition,Applicant has found that following removal of lupeol, betulin andbetulinic acid can be extracted using a solvent comprising carbondioxide at a temperature of about 50° C. to about 120° C. and a pressureof about 5,000 psi to about 10,000 psi.

Accordingly, the processes of the invention can conveniently include afractional supercritical fluid extraction of outer birch bark employinga solvent comprising carbon dioxide at a pressure below about 5,000 psiat a temperature below about 50° C. to provide lupeol followed by anextraction employing a solvent comprising carbon dioxide at a pressureof about 5,000 psi to about 10,000 psi and at a temperature of about 50°C. to about 120° C. to provide betulin and betulinic acid.

The processes of the invention can conveniently include a fractionalsupercritical fluid extraction comprising extracting outer birch barkwith a solvent comprising carbon dioxide for a period of time greaterthan about 30 minutes at a pressure below about 5,000 psi and at atemperature below about 50° C. to provide lupeol followed by extractingwith a solvent comprising carbon dioxide for a period of time greaterthan about 30 minutes at a pressure of about 5,000 psi to about 10,000psi and at a temperature of about 50° C. to about 120° C. to provide amixture of betulin and betulinic acid.

The processes of the invention can conveniently include a fractionalsupercritical fluid extraction comprising extracting with a solventcomprising carbon dioxide for a period of time of about 1 hour to about3 hours at a pressure of about 3,000 psi to about 5,000 psi and at atemperature of about 40° C. to about 50° C. to provide lupeol andbetulin followed by extracting with a solvent comprising carbon dioxidefor a period of time of about 3 hours to about 5 hours at a pressure ofabout 8,000 psi to about 10,000 psi and at a temperature of about 80° C.to about 100° C. to provide a mixture of betulin and betulinic acid.

The processes of the invention can also include non-fractionalsupercritical fluid extraction comprising extracting with a solventcomprising carbon dioxide. The extraction can conveniently be carriedout for a period of time of about 3 hours to about 5 hours at a pressureof about 5,000 psi to about 10,000 psi and at a temperature of about 80°C. to about 100° to provide a mixture of lupeol, betulin and betulinicacid.

Betulin

The betulin from the mixture of lupeol, betulin and/or betulinic acidobtained from supercritical fluid extraction may be purified usingtechniques that are known in the art for purification of naturalproducts, e.g., by recrystallization or by chromatography. Suitablesolvents for crystallization of betulin include, for example, an alcohol(e.g., isopropanol). After purification, betulin is typically at least90% pure. Specifically, betulin is at least 95% pure, or at least 97%pure.

Typically, the processes of the present invention provide betulin in ayield of about 10 wt % to about 25 wt. % based on outer birch bark.Specifically, the yield is about 15 wt % to about 20 wt. % based onouter birch bark, or about 17 wt % to about 20 wt. % based on outerbirch bark.

Using the procedures of the present invention, betulin can be recoveredin about 15 wt. % to about 25 wt. %, based on birch bark. Thisrepresents as much as a 25% improvement over other methods for betulinisolation. O'Connell, M. M.; Bentley, M. D.; Campbell, C. S.; Cole, B.J. W.; Phytochemistry (1988) 27, 2175-2176; Lugemwa, F. N.; Huang, F.Y.; Bentley, M. D.; Mendel, M. J.; Alford, A. R.; J. Agric. Food Chem.,(1990), 36, 493-496.

Betulinic Acid

Betulinic acid may be separated and purified through the specificformation of non-soluble aluminum salts of betulinic acid with aluminumalcoholates. As used herein, “alcoholate” is an organic alcohol whereinthe hydroxy hydrogen has been replaced with a metal, e.g., (CH₃CH₂O)₃Al.Aluminum alcoholates are suitable reagents for triterpene purificationbecause it is believed that aluminum alcoholates bind strongly andirreversibly to acids and tannins, therefore providing completediscoloration of the total extract.

A suitable aluminum alcoholate is aluminum isopropoxide, however, otheralcoholates, basic materials or ion exchange resins may be employed topurify the betulinic acid.

Further purification of betulinic acid from aluminum salts may beprovided using techniques that are known in the art for the purificationor isolation of natural products, e.g., by washing with solvent,crystallization, using ion exchange resins, through the formation ofesters or by chromatography. After purification, betulinic acid istypically at least 90% pure. Specifically, betulinic acid is at least95% pure, or at least 99% pure.

The processes of the present invention yield betulinic acid, afterpurification, that is about 0.5 wt. % to about 2 wt. % based on outerbirch bark. Specifically, betulinic acid is about 1 wt. % to about 1.5wt. % based on outer birch bark, or about 0.5 wt. % to about 1 wt. %based on outer birch bark.

Although betulinic acid is reported as being present in birch bark, theisolation, separation and purification of betulinic acid from birch barkhas not been previously reported. The processes of the present inventionyield betulinic acid of about 0.5 wt. % to 2 wt. % based on the outerbark of betula paparifera. In addition, the processes of the presentinvention may be used to isolate betulinic acid present in other kindsof birch bark. Moreover, the processes of the present invention (e.g.,SCFE) may be used to separate and isolate betulinic acid from otherkinds of plant extracts.

Lupeol

The processes of the present invention yield lupeol, after purification,that is about 0.5 wt % to about 2 wt. % based on outer birch bark.Specifically, the lupeol is about 1 wt % to about 1.5 wt. % based onouter birch bark, or about 0.5 wt % to about 1.0 wt. % based on outerbirch bark.

The lupeol obtained from the supercritical fluid extraction may also bepurified using techniques that are known in the art for purification ofnatural products, e.g., by recrystallization or by chromatography onsilica gel or other suitable supports. Suitable solvents forchromatographic purification of lupeol include, for example, a non-polarsolvent (e.g., hexanes:ether, 4:1). After purification, lupeol istypically at least 90% pure. Specifically, lupeol is at least 95 % pure,or at least 98% pure.

Using the procedures of the present invention, lupeol can be recoveredin about 0.5 wt. % to about 2 wt. %, based on birch bark. Thisrepresents as much as a 70% improvement over other methods for lupeolisolation. O'Connell, M. M.; Bentley, M. D.; Campbell, C. S.; Cole, B.J. W.; Phytochemistry (1988) 27, 2175-2176; Lugemwa, F. N.; Huang, F.Y.; Bentley, M. D.; Mendel, M. J.; Alford, A. R.; J. Agric. Food Chem.,(1990), 36, 493-496.

Applicant has discovered that betulin, lupeol and betulinic acid may beisolated from outer birch bark in good yield and purity, usingsupercritical fluid extraction. Conditions previously known forisolating one or more of these compounds damage much of the suberinpresent in the bark. SCFE, however, is a milder technique which does notdestroy the oxirane rings of suberin.

Isolation of 9,10-Epoxy-18-Hydroxydecanoic Acid (FIG. 4)

As illustrated in FIG. 4, step 1, outer birch bark is subject to alkalihydrolysis. As used herein, “alkali hydrolysis” includes any conditionsuitable for saponifying ester bonds. The reaction (i.e., alkalihydrolysis) can conveniently be carried out in an aqueous alcoholsolution under basic catalysis.

As used herein, “alcohol” is a compound containing at least one C(OH)group. Particular alcohols for use in the present invention will havebetween about 1 and about 10 carbon atoms; may be cyclic or aliphatic;may be saturated or unsaturated; and may be branched orstraight-chained. Specific alcohols suitable for use in the presentinvention include methanol, ethanol, iso-propanol, tert-butanol,1-hepten-3-ol and 1-octen-3-ol.

As used herein, “aqueous alcohol solution” is a solution comprisingwater and an alcohol. Typically, the alcohol is present in at least 20,50, or 75 wt. % of the solution. Specifically, the alcohol is present inabout 90 wt. % of the solution or in about 95 wt. % of the solution. Asillustrated in the Examples herein below, a specific aqueous alcoholsolution suitable for use in the processes of the present invention isabout 5% water in isopropanol.

Suitable bases include metal hydroxides and metal alkoxides. Suitablemetal hydroxides include sodium hydroxide, potassium hydroxide, lithiumhydroxide, calcium hydroxide and barium hydroxide. Suitable metalalkoxides include lithium methoxide, lithium ethoxide, lithiumisopropoxide, lithium tertbutoxide, sodium methoxide, sodium ethoxide,sodium isopropoxide, sodium tertbutoxide, potassium methoxide, potassiumethoxide, potassium isopropoxide, potassium tert-butoxide, magnesiummethoxide, magnesium ethoxide, barium methoxide, barium ethoxide,calcium methoxide and calcium ethoxide.

A specific base suitable for the processes of the present invention issodium hydroxide.

Prior to alkali hydrolysis, outer birch bark can optionally be subjectto extraction as illustrated in FIG. 2 and FIG. 3 to provide extractedouter birch bark. As used herein, “extraction” is the act of obtainingone or more compounds by chemical or mechanical action, as by pressure,distillation, or evaporation as described herein above. Extractionincludes the use of a solvent, for example, water or an organic solvent,at standard temperature and pressure. In addition, extracting alsoincludes supercritical fluid extraction.

Extracting outer birch bark is useful to remove compounds that mayinterfere with the subsequent hydrolysis, or that may contaminate thehydrolysis product. For example, lupeol and betulin are optionallyremoved from the outer birch bark prior to the outer birch bark beingsubject to alkali hydrolysis to facilitate the isolation of9,10-epoxy-18-hydroxyoctadecanoic acid from outer birch bark.

As illustrated in FIG. 4, step 2, the second solution (i.e., aqueousalcohol solution) is separated from the second outer birch bark. Thesolution can be separated using any suitable technique for removing asolid from a liquid. For example, the separation can be accomplished byfiltering, hot filtering, or centrifuging. Specifically, the secondsolution can be separated from the second outer bark by filtering, andmore specifically, by hot filtering. “Hot filtering ” includes filteringa solid from a liquid wherein both the solid and liquid, prior tofiltering, are at a temperature above about 40° C. Specifically, thetemperature is above about 55° C. More specifically, the temperature isabove about 70° C.

As illustrated in FIG. 4, step 3, the second solution is condensed at atemperature below about 50° C. to provide a third solution. The solutioncan be condensed using any suitable technique that is known in the art.For example, “condensing” can include evaporating or evaporating invacuo. Specifically, condensing can occur by evaporating in vacuo at atemperature less than about 50° C. or evaporating in vacuo at atemperature less than about 35° C. More specifically, condensing canoccur by evaporating in vacuo at a temperature less than about 30° C.Condensing can conveniently be carried out for a period of timesufficient to reduce the volume of the solution at least about 20%, 50%,75% or 90%.

Applicant has found that the temperature of condensing (e.g.,evaporation) influences the ability to isolate9,10-epoxy-18-hydroxyoctadecanoic acid from outer birch bark. Applicanthas found that the temperature during condensing preferably should bekept below about 50° C. If the temperature during condensing is keptbelow about 50° C., 9,10-epoxy-18-hydroxyoctadecanoic acid will notreadily decompose to 9,10,18-trihydroxyoctadecanoic acid, and a higheryield of 9,10-epoxy-18-hydroxyoctadecanoic acid can be obtained.

As illustrated in FIG. 4, step 4 and step 5, water, or another suitablesolvent, is added to the third solution to form a precipitate and afourth solution, and the precipitate is separated from the fourthsolution. The precipitate can be separated using any suitable techniqueknown in the art. For example, it may be separated by filtering, hotfiltering, or centrifuging. However, the precipitate typically has aclay-like form. As such, filtration of the precipitate can be extremelydifficult. However, the precipitate can conveniently be separated fromthe fourth solution by centrifuging.

As illustrated in FIG. 4, step 6, the fourth solution is acidified to apH of about 5.5 to about 6.5 to give a fifth solution and9,10-epoxy-18-hydroxydecanoic acid (i.e., oxirane) as a precipitate. ThepH of the fourth solution may be lowered by the addition of a suitableacid. Suitable acids include, for example, hydrochloric acid, phosphoricacid, formic acid, hydrobromic acid, sulfuric acid, nitric acid, aceticacid, and the like.

Typically, the pH should be lowered to a value from about 5.5 to about6.5. If the pH is kept between 5.5 and 6.5, the9,10-epoxy-18-hydroxyoctadecanoic acid will not readily decompose to9,10,18-trihydroxyoctadecanoic acid. In addition, the yield of9,10-epoxy-18-hydroxyoctadecanoic acid decreases if the pH is notcarefully controlled. For example, if the pH falls below 4.0, the9,10-epoxy-18-hydroxyoctadecanoic acid is hydrolyzed to thecorresponding diol in less than two hours at room temperature.

As illustrated in FIG. 4, Step 7, the 9,10-epoxy-18-hydroxyoctadecanoicacid precipitate (i.e., oxirane) is separated from the fifth solution.The the oxirane can be separated from the fifth solution using anysuitable technique. For example, the oxirane can be separated byfiltering, hot filtering, or centrifuging. Specifically, the oxirane canbe separated by filtering, as illustrated in the Examples herein below.

The oxirane can optionally be purified using any suitable techniqueknown in the art. For example, the oxirane can be purified byrecrystallization, extraction, chromatography or sublimation.Specifically, the oxirane can be purified by recrystallization from analcohol (e.g., isopropanol), as illustrated in the Examples hereinbelow.

Isolation of 9,10,18-Tribydroxyoctadecanoic Acid from Outer Birch Bark(FIG. 5)

As illustrated in FIG. 5, step 1, outer birch bark is subject to alkalihydrolysis as described herein above in FIG. 4, step 1.

Lupeol and betulin are optionally removed from the outer birch barkprior to the outer birch bark being subject to alkali hydrolysis tofacilitate the isolation of 9,10,18-trihydroxyoctadecanoic acid fromouter birch bark.

As illustrated in FIG. 5, step 2, the second solution is separated fromthe second outer birch bark as described herein above in FIG. 4, step 2.

As illustrated in FIG. 5, step 3, the second solution is condensed asdescribed hereinabove in FIG. 4, step 3 to form a third solution.

As illustrated in FIG. 5, steps 4 and 5, water is added to the thirdsolution to form a first precipitate and a fourth solution. The firstprecipitate is then separated from the fourth solution as describedherein above in FIG. 4, step 5.

As illustrated in FIG. 5, step 6, the fourth solution is acidified to apH of about 5.5 to about 6.5 as described herein above in FIG. 4, step 6to provide a fifth solution and a second precipitate.

As illustrated in FIG. 5, step 7, the second precipitate is separatedfrom the fifth solution as disclosed herein above in FIG. 4, step 5.

The second precipitate can optionally be crystallized or precipitatedfrom a suitable solvent (e.g., an alcohol) to give a solid and afiltrate and the solid may be separated (e.g., filtered) from thefiltrate.

As illustrated in FIG. 5, step 8, the fifth solution is condensed asdescribed herein above in FIG. 4, step 3 to provide a sixth solution.

As illustrated in FIG. 5, step 9, the sixth solution is subject toepoxidizing conditions to provide an epoxide. The epoxide is thenhydrolyzed. Both reactions can conveniently be carried out in a singlereaction vessel. The sixth solution is subject to epoxidizing conditionsand the resulting epoxide is hydrolyzed under any suitable conditionsknown in the art. E. Seoane and M. Arno, Total Synthesis andStereochemistry of Phloionolic acids, Anales de Quimica, 73, N11,1336-1339 (1977). For example, the sixth solution may be epoxidized andhydrolyzed by the addition of hydrogen peroxide and an acid to the sixthsolution and subsequent heating of the resulting mixture.

As illustrated in FIG. 5, Step 10, 9,10,18-trihydroxyoctadecanoic acidis crystallized from the seventh solution. The crystalline product isthen separated from solution. The crystalline product may be separatedfrom the seventh solution using any suitable technique known in the art.For example, the crystalline product can be separated from solution byfiltering, hot filtering or centrifuging. Specifically, the9,10,18-trihydroxyoctadecanoic acid is separated by filtering.

The 9,10,18-trihydroxyoctadecanoic acid can optionally be purified usingany suitable technique known in the art. For example,9,10,18-trihydroxyoctadecanoic acid can be purified byrecrystallization, extraction, chromatography or sublimation.Specifically, 9,10,18-trihydroxyoctadecanoic acid can be purified byrecrystallization from an aqueous alcohol solution (e.g., ethanol:water,90:10).

Isolation of Non-Soluble Polyphenolic Polymers and Fatty Acids (FIG. 6)

As used herein, “non-soluble polyphenolic polymers” are polymers, whichare non-soluble in water at a pH below about 4.0, but are typicallysoluble in water at a pH above about 6.0. In addition, non-solublepolyphenolic polymers are soluble in acetone, alcohols and other polarsolvents.

As illustrated in FIG. 6, step 1, outer birch bark is subject to alkalihydrolysis as described herein above in FIG. 4, step 1 to provide asecond birch bark and a second solution.

Lupeol and betulin are optionally removed from the outer birch barkprior to the outer birch bark being subject to alkali hydrolysis tofacilitate the isolation of non-soluble polyphenolic polymers and fattyacids from outer birch bark.

As illustrated in FIG. 6, step 2, the second solution is separated fromthe second outer birch bark as described herein above in FIG. 4, step 2.

As illustrated in FIG. 6, step 3, water is added to the second outerbirch bark to provide a third solution and a third outer birch bark.

As illustrated in FIG. 6, step 4, the third outer birch bark isseparated from the third solution as described herein above in FIG. 4,step 5.

As illustrated in FIG. 6, step 5, the third solution is acidified to apH of about 3.0 to about 4.0 to give a fourth solution and a mixture ofnon-soluble polyphenolic polymers and fatty acids (hereinafter “NPPP”).The pH of the solvent may be lowered by adding a suitable acid.Acceptable acids include, for example, hydrochloric acid, sulfuric acid,phosphoric acid, formic acid, hydrobromic acid, nitric acid, acetic acidand the like.

As illustrated in FIG. 6, step 6, the mixture of non-solublepolyphenolic polymers and fatty acids is separated from the fourthsolution as disclosed herein above in FIG. 5, step 10.

The mixture of fatty acids and non-soluble polyphenolic polymers can bepurified using any technique known in the art. For example, the mixtureof non-soluble polyphenolic polymers and fatty acids can be purified byrecrystallization, extraction, chromatography or sublimation.

Using the methods of the present invention, the yield of fatty acids andnon-soluble polyphenolic polymer obtained is about 20 wt. % to about 40wt. % from the outer birch bark.

Isolation of Soluble Polyphenolic Polymers and Fatty Acids from OuterBirch Bark (FIG. 7)

As used herein, “soluble polyphenolic polymer” are that portion of thepolyphenolic polymer fraction dissolved in the media under a specificset of conditions (e.g., solvent, temperature, pH, ionic strength,etc.). Specifically, soluble polyphenolic polymers are soluble in waterin both acidic and basic conditions.

As illustrated in FIG. 7, step 1, outer birch bark is subject to alkalihydrolysis as decsribed herein above in FIG. 4, step 1 to provide asecond outer birch bark and a second solution.

Lupeol and betulin are optionally removed from the outer birch barkprior to the outer birch bark being subject to alkali hydrolysis tofacilitate the isolation of soluble polyphenolic polymers and fattyacids from outer birch bark.

As illustrated in FIG. 7, Step 2, the second outer birch bark isseparated from the second solution as decribed herein above in FIG. 4,step 2.

As illustrated in FIG. 7, step 3, water is added to the second outerbirch bark as described herein above for FIG. 6, step 3 to provide athird outer birch bark and a third solution.

As illustrated in FIG. 7, step 4, the third solution is separated fromthe third outer birch bark as described herein above in FIG. 7, step 4.

As illustrated in FIG. 7, step 5, the third solution is acidified to apH of about 3.0 to about 4.0 as described herein above in FIG. 6, step 5to give a fourth solution and a solid.

As illustrated in FIG. 7, step 6, the solid is separated from the fourthsolution as disclosed herein above in FIG. 4, step 5.

As illustrated in FIG. 7, Step 7, an alcohol is added to the fourthsolution to provide a fifth solution and a precipitate.

As illustrated in FIG. 7, Step 8, the fifth solution is separated fromthe precipitate as disclosed herein above in FIG. 4, step 5.

As illustrated in FIG. 7, Step 9, the fifth solution is condensed asdescribed herein above in FIG. 4, step 3 to provide a mixture of solublepolyphenolic polymers and fatty acids.

The mixture of soluble polyphenolic polymers and fatty acids canoptionally be purified using any suitable technique known in the art.For example, the mixture of soluble polyphenolic polymers and fattyacids can be purified by recrystallization, extraction, chromatographyor sublimation.

The yield of fatty acids and soluble polyphenolic polymers obtained fromthe processes of the invention is typically about 5 wt. % to about 25wt. % from the outer birch bark. Ideally, the yield of fatty acids andsoluble polyphenolic polymers obtained is about 12 wt. % to about 18 wt.% from the outer birch bark.

The present invention will be described by the following examples. Theexamples are for illustration purposes and do not otherwise limit theinvention.

EXAMPLES

Example 1

Dry method of outer birch bark manufacturing.

Birch bark (20 kg) from a drum debarker was air dried (24 hours, roomtemperature) such that the water content was less than 10% and wassubsequently fed into a YardMan Model 246-648D401 chipper/shredder withan 8 HP gas powered motor. The outer bark shreds and inner bark pieces(combined mass of 19.9 kg) were separated on a wire screen with openingsof ¼-by-¼-inch. Outer bark shreds (approximately 5.0 to 6.9 kg) andinner bark chunks (approximately 13.0 to 14.9 kg) were recovered fromthe screening process. The shredded outer bark was reduced in size forextraction using a 15 HP 3B Junior Hammermill made by Jay BeeManufacturing, Inc.

Example 2

Betulin, betulinic acid and lupeol manufacturing.

Dried outer birch bark shreds (1000 g) were loaded in 5 liter fractionalsupercritical fluid extraction vessel. The fractional supercriticalfluid extraction was conducted by Phasex Corporation, 360 MerrimackStreet, Lawrence, Mass. 01843. The first supercritical fluid extractionwas conducted at 45° C. and 4000 psi for two hours, employing carbondioxide as a solvent. After which time, the first fraction (60 grams)was gathered in a separation vessel. The second supercritical fluidextraction was conducted at 90° C. and 9000 psi for 4 hours, employingcarbon dioxide as a solvent. After which time, the second fraction (150grams) was gathered in a separation vessel.

GC/MS analysis of the first fraction: 33% lupeol, 61% betulin, 2%betulinic acid, 4% other triterpenes and fatty acids.

GC/MS analysis of the second fraction: 2% lupeol, 80% betulin, 13%betulinic acid, 5% other triterpenes and fatty acids.

The first fraction (60 g) was boiled with ethanol (1.2 liters) andaluminum isopropoxide (5 g) was added. The resulting mixture was hotfiltered and the filtrate was cooled at 0° C. for 3 hours. Crystalsformed from solution and were filtered to afford betulin (30 g, greaterthan 90% pure, yield 3% from dry bark).

The above filtrate was purified on silica [eluent: hexane-ether (4:1)]employing column chromatography at atmospheric pressure. Lupeol (17 g,1.7 wt. % on dry bark, greater than 90% pure) and betulin (5 g, greaterthan 90% pure, 0.5 wt. % on dry bark) were obtained.

The second fraction (150 g) was boiled with ethanol (3 liters) andaluminum isopropoxide (20 g) was added. The resulting mixture was hotfiltered and the filtrate was evaporated to a volume 0.7 liters andcooled at 0° C. for 3 hours. Crystals fromed from solution and werefiltered to afford betulin (115 g, greater than 90% pure, yield 11.5%from dry bark).

Total yield: betulin: 3%+0.5%+11.5%=15% (from dry bark)

Total yield: lupeol: 1.7% (from dry bark)

Betulinic Acid Separation

a) The solids obtained from the above hot filtration of the firstfraction, ethanol, and aluminum isopropoxide were combined, washed withhot ethanol, acidified with 2% HCl, filtered, washed with hexane, washedwith hexane—ether (1:1), methylated with dimethylsulfide, purified onsilica [eluent: hexane ether (4:1)] and hydrolyzed in 5% sodiumhydroxide in ethanol to afford betulinic acid (16 g, greater than 95%purity, yield 1.6 wt. % on dry bark).

b) Alternatively, the solids obtained from the above hot filtration ofthe first fraction, ethanol, and aluminum isopropoxide were combined,washed with hot ethanol, acidified with 2% HCl, filtered, washed withhexane, washed with hexane-ether (1:1), washed with methylene chloride,and recrystallized from methanol to provide betulinic acid (14 g,greater than 90% purity, yield 1.4 wt. % on dry bark).

The betulin, betulinic acid and lupeol obtained from the methods of thepresent invention were identical to commercial reagents and wereconfirmed by m.p, IR-, H¹-NMR and C¹³-NMR and GC/MS-spectra (fromAldrich Co., in Catalog 1997: Betulin—#12,376-5, p. 166; BetulinicAcid—#85,505-7; from Sigma Co., in Catalog 1999, Lupeol-L 5632, p. 655).

Example 2

Alkali hydrolysis of birch bark, isolation of 22-hydroxydocosanoicfraction and 9,10-epoxy-18-hydroxyoctadecanoic acid fraction

Outer birch bark (790 g) obtained after supercritical fluid extractionwas added to a solution of NaOH (176 g, 4.4 mol) in 95% isopropanol (8liters) and the mixture was refluxed (1 hour). After hot filtration,isopropanol (3.8 liters) was added to the bark and the mixture wasrefluxed (20 minutes). The reaction mixture was filtered and thefiltrate was evaporated in vacuo at 30° C. (GS/MC sample 1). H₂O (5liters) was added to the condensed residue and the mixture was stirredfor 2 hours at room temperature. The insoluble material was separated bycentrifugation and acidified with 6% HCl to pH =4.7 (GS/MC sample 2) toafford 22-hydroxydocosanoic (90 g). The purity of the22-hydroxydocasanoic by GC/MS analysis is greater than 50%. The watersolution after centrifugation (i.e., supernatant) was acidified with 6%HCl to pH =5.5-6.5 and then 9,10-epoxy-18-hydroxyoctadecanoic acidfraction (179 g) was obtained by filtration. The purity of9,10-epoxy-18-hydroxyoctadecanoic fraction by GC/MS analysis is greaterthan 70%.

Example 3

Recrystallization of 9,10-epoxy-18-hydroxyoctadecanoic acid.9,10-Epoxy-18-hydroxyoctadecanoic acid (179 g) was added to isopropanol(1.7 liters) and the mixture was allowed to reflux until all of the acidwas dissolved. The temperature of the solution was decreased to roomtemperature and precipitation occurred over a period of approximately 5hours. After centrifuging, the solution was evaporated and the resultingsolid was crystallized in isopropanol (1 liter). The combined solidswere dried in vacuo and 9,10-epoxy-18-hydroxyoctadecanoic acid (81 g)was obtained. The purity is greater than 95% (GC/MS analysis).

Example 4

Isolation of 9,10,18-trihydrohyoctadecanoic acid (Pholiolic acid).

The supernatant from Example 3 was evaporated and added to a solution of30% H₂O₂ (164 ml, 1.4 mol H₂O₂) in 95% formic acid (1.3 liters) andstirred for 3 hours at 40° C. The mixture was evaporated in vacuo and asolution of 10% NaOH was added (to pH =9.0). The solution was stirredfor 1 hour at 60° C. and 6% aqueous HCl solution was added dropwise tothe stirring mixture (to pH =5.5). The resulting solids were separatedby centrifugation, and dried in vacuo. The solids were crystallized fromethanol-water (4/1) to afford 9,10,18-trihydrohyoctadecanoic acid (96g). The purity is greater than 95% (GC/MS analysis).

Example 5

Polyphenolic polymer and fatty acids separation.

The remaining bark (57 g) from alkaline hydrolysis in Example 2 wasadded to water (7 liters) and stirred for 2 hours. The remaining solids(112 g) were separated by centrifugation and the aqueous solution wasacidified with 37% HCl (to pH =3.0). The resulting mixture ofnon-soluble polyphenolic polymers (200 g) and fatty acids was isolatedvia centrifugation. The aqueous solution separated during centrifugationwas evaporated to a volume of 1 liter and ethanol (3 liters) was added.The solvent was evaporated and a mixture of fatty acids and solublepolyphenolic polymer (SPPP) was obtained. Yield 105 g 10 wt. %) on drybark.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A process for obtaining a natural product fromouter birch bark comprising subjecting the outer birch bark tosupercritical fluid extraction to provide the natural product, whereinthe supercritical fluid extraction utilizes carbon dioxide as a solvent.2. The process of claim 1 wherein the natural product is betulin,betulinic acid or lupeol.
 3. A process for obtaining lupeol, betulinicacid and betulin from outer birch bark comprising: extracting withcarbon dioxide at a pressure between about 3,000 psi and 10,000 psi andat a temperature between about 50° C. and 100° C. to provide lupeol,betulin and betulinic acid.
 4. A process for obtaining lupeol, betulinicacid and betulin from outer birch bark using fractional supercriticalfluid extraction comprising: extracting with carbon dioxide at apressure below about 5,000 psi and at a temperature below about 50° C.to provide a product comprising lupeol; and extracting with carbondioxide at a pressure of about 5,000 psi to about 10,000 psi and at atemperature of about 50° C. to about 120° C. to provide a productcomprising a mixture of betulin and betulinic acid.
 5. The process ofclaim 4 further comprising separating the betulin from the mixture ofbetulin and betulinic acid.
 6. A process for obtaining lupeol from outerbirch bark comprising: subjecting the outer birch bark to supercriticalfluid extraction with carbon dioxide at a temperature of about 40° C. toabout 50° C. and a pressure of about 3,000 psi to about 5,000 psi for aperiod of time of about 1 hour to about 3 hours to provide the lupeol.